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EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS

 
EXTRACTION and CLARIFICATION
Dr. Mario Lebendiker
mariol@cc.huji.ac.il
Tel: 972-2-6586920

Preparation of Cell Lysates /
Different Disruption Methods

Choice of Buffers
 
Inhibitors

Apoptosis Inhibitors

Cell Division/Cell Cycle/Cell Adhesion Inhibitors

Lipid Signal Inhibitors

Neurobiology/Neurodegeneration Inhibitors

Nitric Oxid/Oxidative Stress Inhibitors

Phosphorilation/Dephosphorilation

DNA removal

Endotoxin removal

Cell Penetration
Detergents and detergent removal

Cyclodextrins

Antifoams
Concentration / Ultrafiltration /
Dialysis /
Desalting columns

Preparation of Cell Lysates / Different Disruption Methods

       GE-HEALTHCARE  Protein Sample Preparation Handbook  (pdf)
            Overview of protein sample preparation
            Sample collection, stabilization, and protein extraction
            Increasing detectability of targeted proteins
            Ensuring compatibility in protein sample preparation workflows

      MICROFLUIDICS  Choosing The Best Cell Disruption Method for Research and Pharmaceutical Production  (pdf)
      NOVAGEN   A comprehensive selection of sample preparation tools for three main areas of protein research: expression proteomics, functional
               proteomics, and structural proteomics   (pdf)    (pdf-II)    
           BugBuster® Protein Extraction Reagents. Extraction of active protein from E. coli without sonication
           BugBuster (primary amine-free) Protein Extraction Reagent.  Is a special formulation of BugBuster designed for applications where primary amines
              would interfere if present in the protein extract, such as protein immobilization or cross-linking.
           ProteoExtract® Complete Bacterial Proteome Extraction Kit (C-PEK) is designed for fast and easy extraction of total proteins from bacteria without
               the need for sonication or precipitation.  (pdf-II)
           ProteoExtract® Partial Bacterial Proteome Extraction Kit (P-PEK) provides efficient and standardized extraction of cellular proteins into four distinct
              partial proteomes. The most soluble proteins are released by mechanical disruption in extraction reagent 1, while extraction reagent 2 releases proteins
              of intermediate solubility. Reagent 3 provides efficient extraction of membrane proteins. SDS buffer solubilizes proteins still insoluble in reagent 3. (pdf-II)
          YeastBuster™ Protein Extraction Reagent. Efficient extraction of protein from yeast and plants without mechanical disruption and enzymatic lysis  (pdf-II)
          NucBuster™ Protein Extraction Kit provides an alternative to the time-intensive and cumbersome traditional methods for preparing nuclear
               extracts from mammalian cells. The entire procedure yields ready-to-use nuclear extract within 30 minutes from start to finish  (pdf-II)
          CytoBuster™ Protein Extraction Reagent is a proprietary formulation of detergents optimized for efficient extraction of soluble proteins from
               mammalian and insect cells.  (pdf-II)
          PhosphoSafe™ Extraction Reagent efficiently extracts cytosolic proteins from mammalian and insect cells while preserving their phosphorylation state.
               This reagent contains the same formula as CytoBuster™ Protein Extraction Reagent (1), but also includes four phosphatase inhibitors: sodium
               fluoride, sodium vanadate, b-glycerophosphate, and sodium pyrophosphate.
 (pdf-II)
          ProteoExtract® Complete Proteome Extraction Kits (C-PEKs) are designed for fast and easy extraction of total proteins from yeast and mammalian
               cells and tissues, without the need for sonication or precipitation. (pdf-II)
          ProteoExtract® Partial Proteome Extraction Kits (P-PEKs) provide efficient and standardized extraction of yeast and mammalian cellular
               proteins  into four distinct partial proteomes. The most soluble proteins are released by mechanical disruption in extraction reagent 1,
               while extraction reagent 2 releases proteins of intermediate solubility. Reagent 3 provides efficient extraction of membrane proteins. SDS
               buffer solubilizes proteins still insoluble in reagent 3.  (pdf-II)
          ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from
               mammalian tissue and adherent and suspension-grown cells. The stepwise extraction delivers four distinct protein fractions from one sample:  (pdf-II)
                • Cytosolic protein fraction
                • Membrane/organelle protein fraction
                • Nuclear protein fraction
                • Cytoskeletal protein fraction
          The ProteoExtract® Native Membrane Protein Extraction Kit (M-PEK) is designed for the isolation of native membrane proteins from mammalian
               cells and tissue. The membrane protein fraction is separated in less than two hours without the need for ultracentrifugation or incubation of
               samples at elevated temperatures. (pdf-II)
          PopCulture® Reagent is a detergent-based concentrate that can be added directly to cultures of E. coli to effectively extract proteins without the
               need for cell harvest. The entire culturing, extraction, and purification process can be performed in the original culture tube or multiwell plate. (pdf-II)
          RoboPop™ Solubility Screening Kit is designed for protein expression–level and solubility screening in a 96-well format and includes all reagents
               and plates necessary to perform parallel processing of 96 bacterial cultures. The kit contains PopCulture® Reagent and Lysonase™ Bioprocessing  
               Reagent for efficient extraction of recombinant proteins from E. coli directly from the culture medium without cell harvest, mechanical disruption,
                or extract clarification.
(pdf-II)
           RoboPop™ Ni-NTA His•Bind® and GST•Bind™ Purification Kits are designed for high-throughput (HT) purification of soluble His•Tag® and 
               GST•Tag™ fusion proteins directly from E. coli cultures. (pdf-II)
           Insect PopCulture® Reagent is a detergent-based lysis reagent specifically formulated for total insect cell culture extraction without the need for
                centrifugation.  (pdf-II)
            Insect RoboPop™ Ni-NTA His•Bind® Purification Kit Filtration-based, 96-well format purification directly from transfected cultures of insect
                cells
 (pdf-II)
       NOVAGEN  BugBaster Bacterial Protein Extraction   (pdf)
       NOVAGEN   Lysonase Bioprocessing Reagent   (pdf)
       NOVAGEN   POP culture Reagent. PopCulture® Reagent is used for extraction of proteins from liquid cultures of E. coli without harvesting the cells. 
            Addition of 0.1 culture volume of PopCulture directly to cells in medium, grown at any scale, efficiently extracts proteins while retaining their biological 
            activity 
(pdf)

       NOVAGEN  Protein Extraction Reagents Application Guide. Gentle, efficient, non-mechanical extraction of soluble proteins from bacteria, yeast, 
            mammalian and insect cells: BugBuster®, YeastBuster™, and CytoBuster™ Protein Extraction Reagents. (pdf)
       PIERCE   B-Per Bacterial Protein Extraction Reagent  (pdf)
       QIAGEN:  Qproteome™ Bacterial Protein Preparation Handbook. For preparation of soluble proteins from bacterial cell cultures  (pdf)
       QIAGEN:  Qproteome Mammalian Protein Prep Kit provides gentle but efficient detergent-based lysis of mammalian cells to deliver a total protein
                         fraction suitable for any downstream application.
  (pdf)

       QIAGEN:  Qproteome Cell Compartment Handbook. For fast and easy subcellular fractionation of intact eukaryotic cells. By sequential addition
                        of different extraction buffers to a cell pellet, proteins in the different cellular compartments can be selectively isolated: cytosolic proteins
                        plasma membranes and compartmentalized organelles, such as nuclei, mitochondria, endoplasmic reticulum (ER), and cytoskeletal
                        proteins.
  (pdf)

       QIAGEN:  Qproteome™ Plasma Membrane Protein Handbook. For fractionation of plasma membrane proteins from adherent cell culture
                `        samples. The kit use a  ligand specific for molecules on the plasma membrane vesicles that allow their elution under native conditions
                         after ligand–vesicle complexes are precipitated using magnetic beads that bind to the ligand.
(pdf)

       ROCHE   Complete Lysis Kits: Rapid Protein Extraction and Simultaneous Protection Against Proteases. The complete Lysis-B (2x), -M, and -Y
                        Kits contain an efficient and gentle Lysis Reagent that consists of non-ionic detergents that enable efficient and gentle lysis of bacterial
                        and insect cells, and the complete Protease Inhibitor Cocktail Tablets. The kits are also available as EDTA-free version.
   (pdf)
       ROCHE   Complete Lysis-B (2×) Reagent set for highly efficient protein extraction from bacteria and insect cells by rapid lysis and concurrent
                        protection  of extracted proteins against a multitude of proteases
 (pdf)
       ROCHE   Complete Lysis-M. Reagent set for highly efficient protein extraction from mammalian cells by rapid lysis and simultaneous protection of
                        extracted proteins against a multitude of proteases
 (pdf)
        ROCHE  
Complete Lysis-Y. Reagent set for highly efficient protein extraction from yeast cells by rapid lysis and concurrent protection of extracted
                        proteins against a multitude of proteases
  (pdf)

       Bacterial Protein Extraction (mini-scale) Sonication

      Bacterial Protein Screen (DDM lysis) - Small affinity binding 

       PROTEIN EXPRESSION FACILITY of THE HEBREW UNIVERSITY Sonication of bacterial samples
 

Mitochondria, Nuclear & Membrane Isolation

      CALBIOCHEM    The Cytosol/Mitochondria Fractionation Kit  is designed for extraction of cellular proteomes from mammalian cells and deliver
            two distinct proteins fractions: a mitochondrial fraction and a cytosolic fraction. The procedure is easy to perform and does not rely upon
            ultracentrifugation. Protein fractions are suitable for many downstream applications including 1D and 2D gel electrophoresis, immunoblotting,
            and apoptotic and signal transduction-related translocation studies.  (pdf)
      G-BIOSCIENCES   FOCUSTM SubCell kit enables the fast and easy enrichment of nuclear, mitochondrial, membrane and cytosolic fractions
           from animal cells. The mitochondrial fraction can be subsequently separated into heavy and light fractions by gradient centrifugation. An additional
          step is included to minimize contaminations of the nuclear fraction by cytoplasmic elements. The majority of mitochondria, isolated with this
          kit, contain intact inner and outer membranes. The kit is suitable for cultured animal cells and can be adapted for animal tissues. (pdf-I)  (pdf-II)
      NOVAGEN   A comprehensive selection of sample preparation tools for three main areas of protein research: expression proteomics, functional
            proteomics, and structural proteomics   (pdf)  
            The NucBuster™ Protein Extraction Kit provides an alternative to the time-intensive and cumbersome traditional methods for preparing nuclear
                extracts from mammalian cells. The entire procedure yields ready-to-use nuclear extract within 30 minutes from start to finish
 (pdf-II)
            ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) is designed for fast and reproducible extraction of subcellular proteomes from
                mammalian tissue and adherent and suspension-grown cells. The stepwise extraction delivers four distinct protein fractions from one sample:
                • Cytosolic protein fraction
                • Membrane/organelle protein fraction
                • Nuclear protein fraction
                • Cytoskeletal protein fraction   (pdf-II)
          The ProteoExtract® Native Membrane Protein Extraction Kit (M-PEK) is designed for the isolation of native membrane proteins from mammalian
                cells and tissue. The membrane protein fraction is separated in less than two hours without the need for ultracentrifugation or incubation of
                samples at elevated temperatures.
(pdf-II)
       PIERCE   Lysosome Enrichment Kit for Tissue and Cultured Cells enables isolation and enrichment for intact lysosomes from crude cell and tissue
                        extracts. The kit provides sufficient reagents for preparing 25 extracts and uses OptiPrep™ Cell Separation Media for the density-based
                        separation of lysosomes from contaminating cell structures. The isolated lysosomes may be used for a number of downstream applications,
                        including 2D/MS for proteomics research, electron microscopy, disease profiling and gene expression, signal transduction, and interaction
                        or localization.
(pdf)

      PIERCE    Mitochondria Isolation Kit for Tissue  (pdf)    Offers two methods for isolation of intact mitochondria from both soft and hard tissues.
                        The first method uses a unique reagent-based procedure that enables simultaneous multi-sample processing. The second method relies on
                        traditional Dounce homogenization for tissue disruption and subsequent isolation of the organelle. Both procedures rely on differential
                        centrifugation to separate the intact mitochondria using a bench-top microcentrifuge and are completed in less than 60 minutes.

       PIERCE   Nuclei Enrichment Kit for Tissue enables isolation of an enriched sample containing intact nuclei from 400-500 mg of either hard or soft
                       tissue. The kit contains OptiPrep™ Cell Separation Media and optimized buffers along with detailed instructions for the enrichment of nuclei
                       with minimal contamination from golgi bodies and endoplasmic reticulum. The isolated intact nuclear sample contains all the nuclear proteins,
                       genetic material and other intact nuclear structures, allowing downstream applications such as enzymatic assays, electron microscopy, Western
                       blotting and 2D/MS analysis.
 (pdf)
       PIERCE   
Peroxisome Enrichment Kit for Tissue enables isolation of an enriched peroxisome sample from hard or soft tissue such as rat liver, kidney
                        and heart tissue. The kit provides sufficient reagents for preparing 25 extracts and uses OptiPrep™ Cell Separation Media for the density-based                                                  separation of peroxisomes with minimal contamination from golgi bodies and endoplasmic reticulum.
 (pdf)
       
PIERCE    Subcellular Protein Fractionation Kit enables stepwise separation and preparation of cytoplasmic, membrane, nuclear soluble, chromatin-bound 
                        and cytoskeletal protein extracts from mammalian cultured cells or tissue. The first reagent added to a cell pellet causes selective cell membrane  
                        permeablization, releasing soluble cytoplasmic contents. The second reagent dissolves plasma, mitochondria and ER/golgi membranes but does 
                        not solubilize nuclear membranes. After recovering the intact nuclei by centrifugation, a third reagent yields the soluble nuclear extract. A second
                        nuclear extraction with micrococcal nuclease is performed to release chromatin-bound nuclear proteins. The recovered insoluble pellet is then
                        extracted with the final reagent to isolate cytoskeletal proteins.   (pdf)
       QIAGEN:
  Qproteome™ Plasma Membrane Protein Handbook. For fractionation of plasma membrane proteins from adherent cell culture
                         samples. The kit use a  ligand specific for molecules on the plasma membrane vesicles that allow their elution under native conditions
                         after ligand–vesicle complexes are precipitated using magnetic beads that bind to the ligand.
(pdf)

       QIAGEN:  Qproteome™ Mitochondria Isolation Handbook. For purification of mitochondria from eukaryotic cells.  (pdf)
       QIAGEN:  Qproteome™ Nuclear Protein Handbook. For isolation of nuclear and nucleic-acid binding proteins from eukaryotic cell lysates
                         (pdf)
    (pdf-II)
       QIAGEN:  Qproteome Cell Compartment Handbook. For fast and easy subcellular fractionation of intact eukaryotic cells. By sequential addition
                        of different extraction buffers to a cell pellet, proteins in the different cellular compartments can be selectively isolated: cytosolic proteins
                        plasma membranes and compartmentalized organelles, such as nuclei, mitochondria, endoplasmic reticulum (ER), and cytoskeletal
                        proteins.
  (pdf)


Choice of Buffers
   GE Healthcare  Recommended buffers for Anion Exchange Chromatography

      GE Healthcare  Recommended buffers for Cation Exchange Chromatography

      GE Healthcare  Recommended Volatile buffers systems for Ion Exchange Chromatography

      AMRESCO Buffers Technical Bulletin  (pdf)

     CALBIOCHEM   Buffers - A guide for the preparation and use of buffers in biological systems  (pdf-II)   (pdf)

     CALBIOCHEM   Table of detergents and buffers  (pdf)

      SIGMA    Useful pH Ranges of Selected Biological Buffers   (pdf)

 

Inhibitors

Apoptosis Inhibitors

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

Cell Division/Cell Cycle/Cell Adhesion Inhibitors

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

Lipid Signal Inhibitors

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

Neurobiology/Neurodegeneration Inhibitors

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

Nitric Oxid/Oxidative Stress Inhibitors

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

Phosphorilation/Dephosphorilation Inhibitors

     CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)   
     G-BIOSCIENCES   PhosphataseArrest™ II is a phosphatase inhibitor cocktail consisting of 5 phosphatase inhibitors that target acid, alkaline and
                        tyrosine phosphatases.  (pdf)    (pdf-II)                                                                     
     NOVAGEN   Phosphatase Inhibitor Cocktail Sets  (pdf)   
     PIERCE: Halt™ Protease and Phosphatase Inhibitor Cocktail  (pdf)
     PIERCE:   Halt™ Phosphatase Inhibitor Cocktail. Protects phosphoproteins from serine/threonine phosphatases as well as protein tyrosine
                       phosphatases (PTPs). The cocktail contains a mixture of four inhibitors of broad specificity, including sodium fluoride, sodium orthovanadate,
                       sodium pyrophosphate and β-glycerophosphate.
 (pdf)
   
PROTEINKINASE   A comprehensive source of kinase signaling activators, substrates, inhibitors and kinase-related products.
    ROCHE   PhosSToP Phosphatase Inhbitor Cocktail Tablets to inhibit a broad range of phosphatases.  (pdf)

Protease Inhibitors

      CALBIOCHEM
 Calbiochem Inhibitor SourceBook  (pdf)
      CALBIOCHEM  Protease Inhibitor (pdf)

      CALBIOCHEM  Protease Inhibitor (pdf)    (pdf-II)
      G-BIOSCIENCES  A handbook & selection guide for inhibitors of protease & phosphatases  (pdf)
     
NOVAGEN  Protease Inhibitor Cocktail Sets (with or without EDTA) for bacterial, fungal and yeast cell extracts  (pdf)

     
PIERCE: Halt™ Protease and Phosphatase Inhibitor Cocktail  (pdf)
      ROCHE  Protease Inhibitor Guide   (pdf-I)   (pdf-II)
      ROCHE  Universal Protease Substrate   (pdf)
      SERVA  Protease Inhibitors   (pdf)  
      SIGMA  
Protease and Phosphatase Inhibitor Cocktails   (pdf)  


Others

      CALBIOCHEM  Calbiochem Inhibitor SourceBook  (pdf)

DNA removal
    NOVAGEN Benzonase Nuclease - Removal of Nucleic Acids (pdf)
        NOVAGEN :   Lysonase Bioprocessing Reagent   (pdf)
        Removal of Nucleic Acids - Different Protocols
       
VIVASCIENCE   Removal of DNA from lysates of E. coli cells and French bean tissue prior to electrophoresis.   (pdf)

Endotoxin removal
      Endotoxin Clearance with Triton X-114 – Selected protocols from the literature

        BIORAD   Affi-Prep® Polymyxin Matrix - Removal of endotoxin molecules from biological and aqueous solutions by
                          affinity chromatography
   (pdf)
        BioDtech    EndoBind-R™ is a DADPA-agaroseconjugated S1 peptide affinity chromatography column. It has been used to remove 
                            endotoxin from water, buffers, and cell culture media. Under optimized conditions, it may also be used to produce endotoxin-free 
                            protein solutions with high recovery. Additionally, the S1 peptide is highly resistant to a wide range of pH’s and ionic strengths 
                            making it suitable for many applications in which traditional chromatography methods, such as ion exchangers, affinity ultrafiltration,
                            and immunoaffinity matrices are not applicable   (pdf)
       CAP CODE  Pyrochrome®  Chromogenic Endotoxin Testing. Can achieve a maximum sensitivity of 0.001 EU/mL  (pdf-I)   (pdf-II)
       CHISSO     Cellufine® ET clean for endotoxin removal. The Cellufine ETclean is poly(ε-lysine) immobilized Cellufine® (cellulose spherical 
                        beads). The beads bind and remove endotoxin from your sample solution. The poly(ε-lysine) is a microbial poly(amino acid) that 
                         consist of 30-35 lysine residues produced by Streptomyces albulus. Operating Guidelines: CIP for endotoxin free columns. 
                         Alkali solution and require time for endotoxin free.  (pdf)  
                         (1) To reduce endotoxin from a sample solution containing acidic protein with pI 4.0-6.5, you can use Cellufine ET clean-S beads 
                          with a small pore size at pH 5-7 and NaCl concentration of 0.1-0.4 M.
                          (2) To reduce endotoxin from a sample solution containing neutral or basic protein with pI 7.0-10.5, you can use Cellufine 
                          ET clean-L beads with a large pore size at pH 7-9 and NaCl concentration of 0.1-0.4 M
        GENSCRIPT   The ToxinEraserTM Endotoxin Removal Resin utilizes immobilized polymixin B to bind and remove pyrogens from solution. 
                          The polymixins are a family of antibiotics that contain a cationic cyclopeptide with a fatty acid chain. Polymixin B neutralizes the
                          biological activity of endotoxins by binding to the lipid A portion of bacterial lipopolysaccharide.  (pdf)
        GENSCRIPT   ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit. A quantitative In Vitro end-point endotoxin test. The method utilizes 
                        a modified Limulus Amebocyte Lysate and a synthetic color producing substrate to detect endotoxin chromogenically. The kit has a 
                        minimum endotoxin detection limit of 0.005 EU/ml and a measurable concentration range of 0.005 to 1 EU/ml.   (pdf)

        HYGLOS   EndoLISA® new method for efficient and reliable determination of endotoxin (LPS) levels (alternative to LAL). Use a 
                        lipopolysaccharide (LPS) specific phage-derived protein. Measurement range: 0.05 - 500 EU/ml. pH range: 4 - 9. 
                        No interference in high salt conditions e.g. NaCl, GdnHCl   (pdf-I)   (pdf-II)    
        HYGLOS  Endo Trap Endotoxin Removal System  (pdf-1)  (pdf-2)    (pdf-3)    
        HYGLOS  EndoTrap® blue and EndoTrap® red. EndoTrap is an affinity matrix for the efficient removal of bacterial endotoxins from solutions.
                         (pdf-I) (pdf-II)
(pdf-III)  
                         Application protocol for a FPLC/HPLC automayed system  (pdf)
        
HYGLOS   Endotoxin removal methods - advantages & disadvantages  (attachment)
        MILLIPORE  Matrex Cellufine Sulfate - For concentration, purification and depyrogenation of Virus, Viral/Microbial Antigen,
                        Heparin binding proteins
   (pdf)
        NORGEN  Endotoxin Removal Kit. Designed for the rapid removal of endotoxins from purified DNA.  (pdf)  (pdf Mini)
        PIERCE:   Detoxi-Gel Endotoxin Removing gel: immobilized polymixin B  (pdf)
        PIERCE:    Pierce® High-Capacity Endotoxin Removal Resin. Porous cellulose beads that have been surface modified with covalently attached,
                         modified ε-poly-L-lysine  (pdf)
        PIERCE:    The Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit is an efficient, quantitative endpoint assay for the detection 
                        of gram-negative bacterial endotoxins. Bacterial endotoxin catalyzes the activation of a proenzyme in the modified Limulus Amebocyte Lysate 
                        (LAL). The activated proenzyme then catalyzes the splitting of p-Nitroaniline (pNA) from the colorless substrate, Ac-Ile-Glu-Ala-Arg-pNA; 
                        the activation rate is proportional to the sample endotoxin concentration. After stopping the reaction, the released pNA is photometrically 
                        measured at 405-410nm. The correlation between absorbance and endotoxin concentration is linear in the 0.1-1.0EU/mL range. The developed 
                        color intensity is proportional to the amount of endotoxin present in the sample and can be calculated using a standard curve.   (pdf)
        Pall           Protein Sample Preparation and Analysis Application Manual: 2.6 Endotoxin Removal  (pdf2)
        VIVASCIENCE  Removal of endotoxin from monoclonal antibodies using Vivapure™ centrifugal ion exchange membrane devices   (pdf) 

        Y. Aida and M. Pabst Removal of endotoxin from protein solutions by phase separation using Triton X-114                                                                                                      Journal of lmmunological Methods, 132 (1990) 191-195  (pdf)
        Anspach, Friedrich Birger
 Review: Endotoxin removal by affinity sorbents  J. Biochem. Biophys. Methods 49 (2001) 665–681   (pdf)
        T. Zimmerman et al.   Simultaneous metal chelate affinity purification and endotoxin clearance of recombinant antibody fragments
                       Journal of Immunological Methods 314 (2006) 67–73  (pdf)
        Z. Chen et al. Efficient production of recombinant IL-21 proteins for pre-clinical studies by a two-step dilution refolding method                                                                       Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.02.017  (pdf)

Detergents
    ANATRACE   How to purify a membrane protein-Technical bulletin  (pdf)
        ANATRACE   Structure of Detergents (pdf)
        ANATRACE   Detergent Properties List  (pdf)  
        ANATRACE   Detergents and their use in membrane protein science. Handbook that introduce the researcher to the physical and chemical
                properties of detergents and describe how these properties relate to detergent function. (pdf)  (pdf-II)
        ANATRACE    Amphipol A8-35  Amphipathic surfactant for maintaining solubilized membrane proteins in detergent-free solutions. Serve as 
                stabilizers of membrane proteins in aqueous solutions. Amphipols can substitute out the detergents used to extract membrane proteins, 
                keeping them soluble in detergent-free aqueous solution while stabilizing them biochemically.
                Applications: Stabilization of native membrane protein structure. Facilitating membrane protein folding /refolding. NMR, etc   (pdf)
        ANATRACE    Neopentyl Glycol (NG) class detergents are revolutionary new amphiphiles which have already shown great utility in membrane 
                protein studies. NG class detergents are a more effective detergent for extracting and solubilizing/ stabilizing proteins, and are particularly 
                beneficial in crystallization due to his revolutionary new architecture that allows the molecule to pack densely when forming a micelle, 
                producing exceptionally low critical micelle concentrations and extreme water solubility  (pdf)

               
Nature Methods"Maltose–neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins” 
                by groups from Jean-Luc Popot, Brian Kobilka, and Samuel H. Gellman labs   (pdf)
        ANATRACE    Application References  (pdf)
        
CALBIOCHEM  Biological Detergents. Guide for solubilization of membrane proteins and selecting tools for detergent removal  (pdf)
        CALBIOCHEM  A guide to the properties and uses of detergents in Biology and Biochemistry   (pdf-I)   (pdf-II)
        CALBIOCHEM  Table of detergents and buffers  (pdf-I)   (pdf-II)
        G-BIOSCIENCES   A Handbook & Selection Guide to Detergents & Detergent Removal . Dye solubilization assay for CMC determination  (pdf)
       
G-BIOSCIENCES   Optimizer blueBALLS™. A simple product and method to determine the optimal detergent concentration. Glass balls 
                        that have been coated with a hydrophobic blue dye that has the same properties as membrane proteins. The solubilization of the dye 
                        only occurs when micelles have been formed.  (pdf)

        
GEBA   SDS removing buffer designed for removing bound-SDS from protein sample using ProteoConD kit.  (pdf)
        GE Healthcare   Membrane Protein Purification Kit. Efficient detergent screening of histidine-tagged membrane proteins. Small aliquots of cell 
                     membranes are solubilized in different detergents followed by rapid purification using His Mag Sepharose™ Ni. The purification step 
                    is performed directly after solubilization using the same detergent. Analysis and evaluation can be performed by a number of methods 
                    such as Western blot, gel filtration, or light scattering.   (pdf)

        JENA BIOSCIENCE  JB Screen Detergents cover all detergents that have been successfully used for the crystallization of membrane proteins.

                        (pdf-1)  (pdf-2)  (pdf-3)
 
       MILLIPORE   Detergent Removal with Amicon® Microcon® and Centricon® Centrifugal Devices  (pdf)  
                        Percentage Removal of different detergents after one spin

        NORGEN  ProteoSpin™ Detergent Clean-Up Kit. Provides a fast and simple procedure for the removal of SDS, Triton® X-100 and other detergents
                        from acidic or basic protein samples. It is designed to remove all types of detergents in protein solutions either in their free form or bound form,
                        as when complexed with the protein. Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation
                        matrix
.  (pdf Micro)  (pdf Maxi)
        PALL       SDR HyperD® Detergent Removal and Solvent removal (e.g. Tri-n-Butyl Phosphate – or TnBP). Stable in acid, polar organic
                        and oxidizing solutions. (pdf-I)  (pdf-II)
        PALL       Protein Sample Preparation and Analysis Application Manual:  2.3 Detergent Removal  (pdf2)
        PIERCE:  Extracti-Gel D Detergent Removing gel   (pdf)
        PIERCE:  Remove detergent from protein samples  (pdf) 
                        Overview of Removal Methods. General Detergent Properties. Appropriate Methods of Removal for Specific Detergents

        SIGMA   Detergents. Detergent Properties and Applications. Detergent Types and Selection. Detergent Selection Table. BioUltra Detergents  
                        (pdf - see pg 13)
        SIGMA   Cyclodextrins  (pdf - see pg 32)
        SIGMA   Antifoams  (pdf - see pg 33)
        SIGMA   Detergent Removal  (pdf - see pg 35
        SIGMA   Detergents Properties and Applications  (pdf)
        SIGMA   Table of detergents  (pdf)

        


     Proteases sensitivity to Detergents

        James M. Vergis, Michael C. Wiener (2011)    The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal
            Protein Expression and Purification 78: 139–142  (pdf)

        Mohanty A. et.al. (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expression and Purification 27: 109–114  (pdf)

Cell Penetration
 

        ABBIOTEC   Lipodin-ProTM is a protein transfection reagent, for transporting biologically active peptides and proteins directly into living cells.  
                Is a lipid-based formulation that is added to the protein of interest minutes before cell delivery. Within few hours, protein activity can be observed 
                in living or fixed cells. Ideal for monitoring kinetics of biological activities in established cell lines and primary cells, including neurons.  No chemical    
                ligation or crosslinking. Serum compatible, no cytotoxicity and biodegradable. Up to 95% efficiency in less than 5 hours. (pdf)
        ABBIOTEC   Lipodin-AbTM is a  reagent optimized for Antibody delivery into living cells. A lipid-based formulation that is added to the antibody 
                solution minutes before cell delivery.  Within few hours, antibody staining can be observed in living or fixed cells. Ideal for staining intracellular proteins 
                without fixation in established cell lines and primary cells, including neurons.  No need for cell fixation protocol. Serum compatible, no cytotoxicity and 
                biodegradable. Deliver functionally active antibody within hours. Up to 95% efficiency.
(pdf)
                 ACTIVE MOTIVE    Chariot™  is a revolutionary delivery reagent that quickly and efficiently transports biologically active proteins, peptides and antibodies 
                directly into cells. The typical delivery efficiency is 60-95%. Less than two hours after delivery, live cells can be assayed to determine the effects of the 
                introduced material, without the need for fixing. This makes Chariot an ideal tool for functional studies, including delivering inhibitory proteins, labeling 
                organelles, screening peptide libraries and studying protein half-lives & transient complementation.  (pdf)
    JENA BIOSCIENCE  “Protein Transduction Domains” (PTDs) or “Cell penetrating peptides” (CPPs) used to internalize peptides, proteins,
                    oligonucleotides, nucleic acids, modified nucleic acids like PNAs or morpholino oligonucleotides and plasmids. CPPs were successfully 
                    tested on a large number of different cells including mammalian cells and plant cells, cell cultures and primary cells; intracellular 
                    delivery into nerve cells and for penetration of the blood-brain barrier.  (pdf-I)  (pdf-II)  
                        JBS-Proteoducin:  Cocktail developed for internalization of peptides and proteins into living cells
                        JBS-Nucleoducin: Internalization of mono- and olgonucleotides into live cells
                        The amphiphilic CPPs MPG , MPG and CAD-2 are designed for formation of non-covalent complexes with peptides, proteins, 
                            plasmids and nucleic acids and their mimics. They strongly differs in their polarities.
                        Penetratin and HIV-Tat are classical CPPs that are widely used for forming conjugates or fusion proteins    

Concentration / Ultrafiltration / Dialysis / Desalting Columns

    Protein concentration by ultrafiltration and buffer solubility screen
    AmiKa Corp   Produce a unique range of products for microdialysis of small (10 to 5000 microlitres) sample volumes (proteins, peptides, nucleic acids
                        and other biomolecules), electroelution,  electrodialysis, electro-concentration, Coomassie Blue removal, and quantitative protein determination
        BioToolomics:    SuperSpinTM Desaltor    (pdf)
        GE Healthcare    Selecting. Hollow fiber. Cartriges and Systems   (pdf)
        
GE Healthcare    Membrane separations: range of options   (pdf)   
        GE Healthcare    PD-10 Desalting Columns, PD MidiTrap G-25, PD MiniTrap G-25, PD SpinTrap G-25, and PD MultiTrap G-25.
                        Prepacked, single-use columns and 96-well filter plates for a wide range of applications including desalting, buffer exchange, and the
                        removal of low-molecular weight compounds. Centrifugation protocol with all gravity columns.  (pdf)
       GE Healthcare   PD 10 Desalting columns  (pdf)    
       GE Healthcare   PD SpinTrap G-25  (pdf)
 
      GE Healthcare   Vivaspin 500, 2, 6 and 20.  Vivaspin Concentrators are disposable ultrafiltration devices for the concentration of biological samples (pdf)
       G-BIOSCIENCES    The Column PROTEIN- Concentrate Kit has been specifically developed for concentration of those proteins that cannot be
                        concentrated either by precipitation or other techniques. The kit is based on a proprietary Protein Binding Resin that binds and immobilizes
                        any protein in low salt buffer between pH 2-12 (capacity ~ 0.5mg protein/ml Protein Binding Resin). The immobilized protein is spin-eluted
                        in a small volume of specifically formulated elution buffer, giving several fold effective concentration. This kit is suitable for concentration of
                        a total of 4mg protein in either single or multiple procedures.  (pdf)
       G-BIOSCIENCES    The OrgoSol PROTEIN Concentration™ kit is based on precipitation of protein in a proprietary combination of organic
                            solvents. The kit has been specifically developed for efficient precipitation of protein solution with a minimal disruption to the protein
                            structure and thus maintaining biological activity of most proteins. This kit is therefore suitable for concentration of protein solutions
                            where maintaining biological activity is essential.  (pdf)
       G-BIOSCIENCES     Tube-O-DIALYZER™   (pdf) 
       
GEBA   Maxi GeBAflex-tube Gel Extraction and Dialysis Kit (3 ml) Handbook. Applications: Extraction of proteins, RNA, DNA or oligonucleotides             
                            from polyacrylamide, agrose or any gel matrix in any running buffer; dialysis or buffer exchange of volumes between 0.1-3 ml;
                            and preparation of protein  samples for MALDI-MS.
(pdf-I)  (pdf-II)

        MILLIPORE - AMICON: Protein Purification, Concentration and Dialysis  (pdf)   (pdf-II)
        MILLIPORE   Stirred Ultrafiltration Cells Models 8003, 8010, 8050, 8200, 8400. User Guide. Millipore stirred ultrafiltration cells are designed for
                                rapid concentration or purification of macromolecular solutions in volumes from 3 to 400 mL
 (pdf)

        MILLIPORE   Solvent Resistant Stirred Cells for Ultrafiltration and Filtration Applications  (pdf)
        MILLIPORE   Model 2000 High-performance Ultrafiltration Cell. The 2000 series of internally stirred ultrafiltration cells are designed for rapid  
                                concentration or purification of macromolecular solutions in volumes up to 2000 ml.  (pdf)
        MILLIPORE - AMICON ( Ultrafiltration) Centrifuge Filter Devices  (pdf)
        MILLIPORE   Detergent Removal with Amicon® Microcon® and Centricon® Centrifugal Devices  (pdf)  
                               Percentage Removal of different detergents after one spin

       
MILLIPORE    Membrane-based Tangential Flow Filtration (TFF). Membrane-based Tangential Flow Filtration (TFF) unit operations are used
                                for clarifying, concentrating, and purifying proteins. This technical brief is a practical introduction to protein processing using tangential
                                flow filtration. It is intended to help scientists and engineers achieve their protein processing objectives by discussing how the choice
                                of key components and operating parameters will affect process performance.  (pdf)
        MILLIPORE    Ultrafiltration Membranes for Macromolecule Processing . This guide provides an introduction to Millipore’s family of Ultrafiltration    
                                Membranes and an overview of the key criteria and product characteristics to consider when selecting an UF membrane  (pdf)

        NORGEN   ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Kit. The simultaneous removal of salts while concentrating a dilute
                           protein solution makes the kit a convenient method for preparing proteins before running many downstream applications such as SDS-PAGE
                           and isoelectric focusing. Purification is based on spin column chromatography using Norgen’s proprietary resin as the separation matrix. These
                           large columns are frequently used to prepare protein samples for structural analysis where larger amounts of protein are needed, such as
                           X-ray crystallography, NMR spectroscopy and other applications. The ProteoSpin™ CBED Maxi Kit comes with solutions for concentrating
                           and desalting both acidic and basic proteins.
 (pdf for Micro)  (pdf for Maxi)

        NOVAGEN  D-Tube™ Dialyzers and D-Tube Electroelution Kit . Easy-to-handle dialyzers in a capped centrifuge tube format with dialysis membrane
                            windows for buffer exchange and removal of solutes. The disposable, single-use tubes require no syringes, centrifuge, or laborious steps
                            to manipulate small sample volumes. Available with molecular weight cut-offs from 3.5 to 14 kDa and are designed with three volume
                            capacities: mini (10–250 ml), midi (50–800 ml), and maxi (100–3000 ml). The membrane is ultra-clean, EDTA-treated regenerated
                            cellulose, sulfur- and heavy metal-free.  (pdf)
         NOVAGEN  D-Tube96 Dialyzer. The membrane is ultra clean, EDTA-treated regenerated cellulose, sulfur- and heavy metal-free and available in
                            molecular weight cutoffs of 6-8 kDa or 12-14 kDa. The D-Tube Dialyzers Mini have a capacity of 10-250 μl and are ideal for small
                            samples.   (pdf)
        
Pall         Protein Sample Preparation and Analysis Application Manual:
                            2.4 Concentration, Desalting, and Buffer Exchange (pdf2)

                            4.4 Buffer Exchange, Desalting, and Concentration  (pdf4)
                            6.2 Optional Pre-Treatment to Improve Recovery of Samples from Ultrafiltration Spin Filters  (pdf6)
                            6.4 General Filtration Procedures for AcroPrep™ and AcroWell™ Filter Plates  (pdf6)
        Pall         Centrifugal Devices for Ultrafiltration & Microfiltration. 
A full range of precise, high-performance products to rapidly process volumes from
                        50 µL to 60 mL.
 (pdf-I)    (pdf-II)     (pdf-III)                                                                                                                               
        Pall         Trisacryl® GF05 M, LS Size Exclusion Chromatography Sorbents. Designed for desalting and for the separation of small molecules.
                        40-80 μm for M grade & 80-160 μm for LS grade for fractionation at high flow with low pressure, designed for industrial scale.  (pdf)
       
Pall          Products for Sterile Filtration, Clarification and Separation in the Laboratory. Technologies for Complex Lab Bioprocessing   (pdf)
        
PIERCE    iCON™ Concentrator 7 ml/9K, 7 ml/20K, 20mlml/9K and 20mlml/20K . Disposable ultrafiltration centrifugal devices for concentration
                         and diafiltration/buffer exchange of biological samples such as enzymes, antibodies or DNA.
 (pdf-I)  (pdf-II)
        PIERCE    Protein desalting Spin Columns   (pdf)
        PIERCE    Zeba™ 96-well Desalt Spin Plates contain a resin that offers high-performance desalting and protein recovery characteristics for
                          multiple sample processing. Sample volumes from 20 to 100 μl can be processed with > 95% retention of salts and other small
                          molecules (< 1,000 Da) and exceptional protein recovery.
(pdf)  
        PIERCE   Zeba™ Micro Desalt Spin Columns  (pdf)
        PIERCE   High-Performance Dialysis and Desalting Brochure: Slide-A-Lyzer® MINI Dialysis Units; iCON™ Protein Concentrators;
                        SnakeSkin® Dialysis Tubing and Desalting Columns  (pdf-I)   (pdf-II)
        PIERCE   Dyalisis Cassettes  (pdf)
        PIERCE   MINI Dyalisis Unit   (pdf)
        SARTORIUS (Ultrafiltration) Centrifugal Units - Centrisart Units. Spiral Flow Filtration Cells (10-100ml)
        SPECTRUM LABORATORIES  Float-A-Lyzer® • DispoDialyzer® • Micro DispoDialyzer® Dialysis Membranes with Different Molecular
                        Weight Cut-offs   (pdf)
 (pdf-II)  
                        Tangential flow membrane for Biomedical Separation

        THE NEST GROUP Ultra-filtration, Dialysis, Spin Columns or 96 Well Plates for Salt Removal - Buffer Exchange
                          Salt Removal - Buffer Exchange MicroDialysis, Equilibrium Dialysis, and Electroelution. Single-Sided Biodialyzer, Double-Sided
                                Biodialyzer, Disposable Dialyzers, Equilibrium Dialysis and Membranes and Electroelution  (pdf)
                           Equilibrium dialysis  (pdf)
                           96-Well DispoDIALYZER™- Samples from 25μl to 300μl. Range of membrane MWCO’s available: 1K, 2K, 5K, 10K,
                                and 25K Daltons  (pdf)   
                           Micro DispoDialyzer™ - Samples from 5μl to 100μl and Macro Fast DispoDIALYZER™ - Samples from 1ml to 10ml   (pdf )
        VIVASCIENCE Ultrafiltration Products Catalogue  (pdf-I)  (pdf-II)   (pdf-III)
        VIVASCIENCE  Vivapure® C18 Micro spin columns. Innovative membrane-based devices for concentration, purification and desalting of peptides
                                    prior to analysis by mass spectrometry.
(pdf-I) (pdf-II) (pdf-III)
        VIVASCIENCE  Concentration of Peptides with Vivaspin 500 Ultrafiltration Devices  (pdf)



Entries Since September 2006  


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Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@cc.huji.ac.il  Tel: 972-2-6586920  

 

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